Figure 4. AAV replication in yeast.

(A) Southern blot analysis of low M_r DNA of two different yeast clones URA3⁺ (lane 1 and 2) derived from transformation with pAAVRepURA3 using the URA3 marker gene as probe. (B, C) Southern blot analysis of genomic DNA of the same two clones as in B undigested (lane 1 and 3), and digested with AseI (lane 2 and 4) probed with URA3 (B) or ITR probe (C). (D) Low M_r DNA of the two yeast clones URA3⁺LEU2⁺ derived from transformation with pAAVRepURA3 and successively transformed with pG.Rep68 (lane 1 and 2) analyzed on Southern Blot using the URA3 probe. (E) Low M_r DNA of yeast clones URA3⁺LEU2⁺ derived from co-transformation of pAAVRepURA with control plasmid pGAD424. Lanes 1 and 3 show the undigested DNA and lanes 2 and 4, DNA digested with DpnI and subjected to Southern blot analysis using URA3 marker gene as probe. Lane 5 shows the result of DpnI digestion of the pAAVRepURA plasmid. DpnI was performed in order to demonstrate that AAV DNA replicated in yeast. (F) Low M_r DNA of yeast clones URA3⁺LEU2⁺ derived from co-transformation of pAAVRepURA with plasmid pG.Rep68. Lanes 1, 3, 5, 7 show the undigested DNA and lanes 2, 4, 6, 8 DNA digested with DpnI and subjected to Southern blot analysis using URA3 marker gene as probe. (G) Schematic representation of DpnI/MboI (indicated with D) and AseI (indicated with A) restriction map of pAAVRepURA plasmid. The DpnI/MboI restriction endonucleases do not cut in the URA3 gene.